Autophagy-Inducing Peptide Analogs

ABSTRACT

An autophagy-inducing compound comprises an autophagy-inducing peptide comprising Beclin 1 peptides immediately N- and C-terminally flanked by moieties R 1  and R 2 , respectively, wherein up to six of said peptide residues may be substituted, R 1  and R 2  do not naturally flank the Belclin 1 residues, and F270 and F274 are optionally substituted and optionally linked. The compounds may be used to induce autophagy.

This is a continuation of PCT/US14/18467, filed Feb 26, 2014, whichclaims priority to Ser No. 61/803,095, filed Mar 18, 2013.

This invention was made with government support under Grant NumberU54AI057156 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

INTRODUCTION

Beclin 1 encodes a 450 amino acid protein with a central coiled coildomain. Within its N′ terminus, it contains a BH3-only domain, whichmediates binding to anti-apoptotic molecules such as Bcl-2 and Bcl-xL.The most highly conserved region, referred to as the evolutionarilyconserved domain (ECD), spans from amino acids 244-337, which isimportant for its interaction with Vps34. Overexpression of Beclin 1 issufficient to induce autophagy. Furuya, et al., Autophagy 1, 46-52,2005; Pattingre, S. et al. Cell 122, 927-39, 2005.

We disclosed an autophagy-inducing beclin 1 peptide in PCT/US13/22350.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for inducing autophagy,including compounds comprising no more than a 9, 10 or 11-mer Beclin 1peptide (from residue 269 or 270 to residue 278 or 279).

In one aspect the invention is an autophagy-inducing compound comprisingan autophagy-inducing peptide comprising Beclin 1 residue 269 or 279 toBeclin 1 residue 278 or 279, wherein Beclin 1 residues 269-279 isVFNATFHIWHS (SEQ ID NO:01), or the D-retro-inverso sequence thereof,wherein the peptide is immediately N- and C-terminally flanked bymoieties R₁ and R₂, respectively, wherein up to six of said residues maybe substituted, R₁ and R₂ do not naturally flank the Beclin 1 peptide,and F270 and F274 are optionally substituted and optionally linked.

In particular embodiments the invention is a subject autophagy-inducingcompound,

-   -   having a peptide sequence disclosed herein, unsubstituted, or        wherein up to six of said residues may be substituted, and the        two F residues are F₁ and F₂ and are optionally substituted and        optionally linked, or the D-retro-inverso sequence of the        peptide;    -   wherein R₁ is a heterologous moiety that promotes therapeutic        stability or delivery of the compound;    -   wherein R1 comprises a transduction domain, a homing peptide, or        a serum stabilizing agent;    -   wherein R1 is a tat protein transduction domain linked to the        peptide through a diglycine linker, particularly a diglycine-T-N        linker;    -   wherein R₂ is carboxyl;    -   wherein R₂ comprises an affinity tag or detectable label,        particularly a fluorescent label;    -   wherein F270 and F274 are substituted and linked;    -   wherein F270 and F274 are substituted with crosslinkable        moieties and/or linked, and each optionally comprises an        additional a-carbon substitution selected from substituted,        optionally hetero- lower alkyl, particularly optionally        substituted, optionally hetero- methyl, ethyl, propyl and butyl;    -   wherein F270 and F274 are substituted with homocysteines        connected through a disulfide bridge to generate a ring and tail        cyclic peptide;

wherein the side chains of F270 and F274 are replaced by a linker:

-   -   —(CH₂)_(n)ONHCOX(CH₂)_(m)—, wherein X is CH₂, NH or O, and m and        n are integers 1-4, forming a lactam peptide;

—CH₂OCH₂CHCHCH₂OCH₂—, forming an ether peptide; or

-   -   —(CH₂)_(n)CHCH(CH₂)_(m)—, forming a stapled peptide;    -   wherein 1 to 6 residues are alanine substituted;    -   wherein the peptide comprises at least one of substitutions:        H275E and S279D.    -   wherein the peptide comprises one or more D-amino acids,        L-P-homo amino acids, D-β-homo amino acids, or N-methylated        amino acids;    -   comprising the D-retro-inverso sequence, particularly,        RRQRRKKKRGY-GG-DHWIEFTANFV (SEQ ID NO:08);    -   wherein the peptide is acetylated, acylated, formylated,        amidated, phosphorylated, sulfated or glycosylated;    -   comprising an N-terminal acetyl, formyl, myristoyl, palmitoyl,        carboxyl or 2-furosyl group, and/or a C-terminal hydroxyl,        amide, ester or thioester group; and/or    -   wherein the peptide is cyclized.

The invention also provides pharmaceutical compositions comprising asubject compound in unit dosage, administrable form, and methods ofinducing autophagy, comprising administering to a person in need thereofan effective amount of a subject compound or composition.

The invention includes all combinations of the recited particularembodiments as if each combination had been laboriously separatelyrecited.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

In one aspect the invention is an autophagy-inducing compound comprisingan autophagy-inducing peptide comprising Beclin 1 residues 270-278(FNATFHIWH; SEQ ID NO:01, residues 2-10), or the D-retro-inversosequence thereof, immediately N- and C-terminally flanked by moieties R₁and R₂, respectively, wherein up to six of said residues may besubstituted, R₁ and R₂ do not naturally flank the Beclin 1 residues, andF270 and F274 are optionally substituted and optionally linked.

In embodiments the peptide comprises an unsubstituted mutant sequence ofTable 1, below, or the D-retro-inverso sequence thereof, immediately N-and C-terminally flanked by moieties R₁ and R₂, respectively, wherein R₁and R₂ do not naturally flank the Beclin 1 residues.

Peptide and compound activity are tolerant to a variety of additionalmoieties, flanking residues, and substitutions within the definedboundaries. Peptide and compound activity are also tolerant to backbonemodification and replacement, side-chain modifications, and N- andC-terminal modifications, all conventional in the art of peptidechemistry.

Chemical modifications of the peptide bonds may be used to provideincreased metabolic stability against enzyme-mediated hydrolysis; forexample, peptide bond replacements (peptide surrogates), such astrifluoroethylamines, can provide metabolically more stable andbiologically active peptidomimetics.

Modifications to constrain the peptide backbone include, for example,cyclic peptides/peptidomimetics which can exhibit enhanced metabolicstability against exopeptidases due to protected C- and N-terminal ends.Suitable techniques for cyclization include Cys-Cys disulfide bridges,peptide macrolactam, peptide thioether, parallel and anti-parallelcyclic dimers, etc.; see, e.g. PMID 22230563 (stapled peptides), PMID23064223 (use of click variants for peptide cyclization), PMID 23133740(optimizing PK properties of cyclic peptides: effects of side chainsubstitutions), PMID: 22737969 (identification of key backbone motifsfor intestinal permeability, PMID 12646037 (cyclization by coupling2-amino-d,l-dodecanoic acid (Laa) to the N terminus (LaaMII), and byreplacing Asn with this lipoamino acid).

In particular embodiments F270 and F274 are substituted and linked, suchas wherein the side chains of F270 and F274 replaced by a linker. Forexample, these residues may be substituted with homocysteines connectedthrough a disulfide bridge to generate a ring and tail cyclic peptide.In addition, the side chains of these residues can be substituted andcross-linked to form a linker, such as —CH₂)_(n)ONHCOX(CH₂)_(m)—,wherein X is CH₂, NH or O, and m and n are integers 1-4, forming alactam peptide; —CH₂OCH₂CHCHCH₂OCH₂—, forming an ether peptide;—(CH₂)_(n)CHCH(CH₂)_(m)—, forming a stapled peptide. The linkers mayincorporate additional atoms, heteroatoms, or other functionalities, andare typically generated from reactive side chain at F270 and F274. Thecrosslinkable moieties may include additional α-carbon substititions,such as optionally substituted, optionally hetero- lower alkyl,particularly optionally substituted, optionally hetero- methyl, ethyl,propyl and butyl. Exemplary crosslinkable moieties include:

Other suitable modifications include acetylation, acylation (e.g.lipopeptides), formylation, amidation, phosphorylation (on Ser, Thrand/or Tyr), etc. which can be used to improve peptide bioavailabilityand/or activity, glycosylation, sulfonation, incorporation of chelators(e.g. DOTA, DPTA), etc. PEGylation can be used to increase peptidesolubility, bioavailability, in vivo stability and/or decreaseimmunogenicity, and includes a variety of different PEGs: HiPEG,branched and forked PEGs, releasable PEGs; heterobifunctional PEG (withendgroup N-Hydroxysuccinimide (NHS) esters, maleimide, vinyl sulfone,pyridyl disulfide, amines, and carboxylic acids), etc.

Suitable terminal modifications include N-terminal acetyl, formyl,myristoyl, palmitoyl, carboxyl and 2-furosyl, and C-terminal hydroxyl,amide, ester and thioester groups, which can make the peptide moreclosely mimic the charge state in the native protein, and/or make itmore stable to degradation from exopeptidases.

The peptides may also contain atypical or unnatural amino acids,including D-amino acids, L-β-homo amino acids, D-β-homo amino acids,N-methylated amino acids, etc.

A wide variety of flanking moieties R₁ and/or R₂ may be employed, suchas affinity tags, transduction domains, homing or targeting moieties,labels, or other functional groups, such as to improve bioavailabilityand/or activity, and/or provide additional properties.

One useful class of such moieties include transduction domains whichfacilitate cellular penetrance or uptake, such as protein-derived (e.g.tat, smac, pen, pVEC, bPrPp, PIs1, VP22, M918, pep-3); chimeric (e.g.TP, TP10, MPGΔ) or synthetic (e.g. MAP, Pep-1, Oligo Arg)cell-penetrating peptides; see, e.g. “Peptides as Drugs: Discovery andDevelopment”, Ed. Bernd Groner, 2009 WILEY-VCH Verlag GmbH & Co, KGaA,Weinheim, esp. Chap 7: “The Internalization Mechanisms and Bioactivityof the Cell-Penetrating Peptides”, Mats Hansen, Elo Eriste, and UloLangel, pp. 125-144.

Another class are homing biomolecules, such as RGD-4C (CCDCRGDCFC; SEQID NO:04), NGR (CCNGRC; SEQ ID NO:05) , CREKA, LyP-1 (CGNKRTRGC; SEQ IDNO:06), F3, SMS (SMSIARL; SEQ ID NO:07), IF7, and H2009.1 (Li et al.Bioorg Med Chem. 2011 Sep 15;19(18):5480-9), particularly cancer cellhoming or targeting biomolecules, wherein suitable examples are known inthe art, e.g. e.g. Homing peptides as targeted delivery vehicles, PirjoLaakkonen and Kirsi Vuorinen, Integr. Biol., 2010, 2, 326-337; Mappingof Vascular ZIP Codes by Phage Display, Teesalu T, Sugahara K N,Ruoslahti E., Methods Enzymol. 2012;503:35-56.

Other useful classes of such moieties include stabilizing agents, suchas PEG, oligo-N-methoxyethylglycine (NMEG), albumin, an albumin-bindingprotein, or an immunoglobulin Fc domain; affinity tags, such asimmuno-tags, biotin, lectins, chelators, etc.; labels, such as opticaltags (e.g. Au particles, nanodots), chelated lanthanides, fluorescentdyes (e.g. FITC, FAM, rhodamines), FRET acceptor/donors, etc.

The moieties, tags and functional groups may be coupled to the peptidethrough linkers or spacers known in the art, such as polyglycine,ε-aminocaproic, etc.

The compound and/or peptide can also be presented as latent oractivatable forms, such as a prodrug, wherein the active peptide ismetabolically liberated; for example, release of the linear peptide fromcyclic prodrugs prepared with an acyloxyalkoxy promoiety (prodrug 1) ora 3-(2′-hydroxy-4′,6′-dimethylphenyl)-3,3-dimethyl propionic acidpromoiety (prodrug 2) of the compound).

Particular embodiments include all combinations of particularembodiments, as though each had be separately set forth; for example,wherein the peptide is N-terminally flanked with T-N and C-terminallyflanked by T, the first moiety is a tat protein transduction domainlinked to the peptide through a diglycine linker; and wherein thepeptide is N-terminally flanked with T-N and C-terminally flanked by T,the first moiety is a tetrameric integrin α(v)β(6)-binding peptide knownas H2009.1, linked to the peptide through a maleimide—PEG(3) linker.

In another aspect the invention provides a method of inducing autophagy,comprising administering to a person in need thereof an effective amountof a subject compound or composition. Applications broadly encompasspersons in need of enhanced autophagy, and include diseases andpathologies where the upregulation of autophagy is therapeuticallybeneficial, including infection with intracellular pathogens,neurodegenerative diseases, cancers, cardiomyopathy, and aging.

Autophagy can be detected directly, indirectly or inferentially byconventional assays, such as disclosed and/or exemplified herein,including biochemically (by assessing the generation of Atg8-PE orLC3-II or the degradation of p62) or microscopically (e.g. by observingthe localization pattern of fluorescently tagged Atg8 or LC3).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein, including citations therein, are herebyincorporated by reference in their entirety for all purposes.

EXAMPLES

We used the beclin 1 ECD structure as a basis for rational design andoptimizing the beclin 1 peptide as a therapeutic.

A sequence alignment of Atg6/beclin 1 orthologues revealed two conservedaromatic residues (F270 and F274) within the Tat-beclin 1 peptide thatare required for its autophagy-inducing activity [1]. When mapped to thebeclin 1 ECD structure, these aromatic side chains interact in aT-shaped geometry and help to position the intervening residues in asurface-exposed loop. We inferred that the aromatic residues play ananalogous role in the Tat-beclin 1 peptide, thus contributing to itsstructural stability, and that replacing this aromatic interaction witha more permanent chemical linkage should further improve conformationalstability [2]. For example, the two aromatics could be replaced withhomocysteines that exhibit a similar length as the native sidechains andcan be cyclized to form a stabilized ring-and-tail peptide structure.Similar biologically active cyclic peptide structures exist in nature,such as the 6-amino acid ring and 3 amino acid tail structure of thepeptide hormone vasopressin [3] or the 8-amino acid ring and 13 aminoacid tail structure of the RNA polymerase inhibiting peptide microcin[4].

To further assess the components of the beclin 1 peptide required forautophagy-inducing activity, a series of shorter peptides weredeveloped. Activity was measured by biochemical autophagy assaysincluding western blot detection of p62 (a protein degraded byautophagy) and western blot detection of the conversion of LC3-I intoLC3-II (the lipidated autophagosome-associated form of LC3). 10micomolar concentration of Tat-GG-VFNATFEIWHD (SEQ ID NO:09) results inincreased p62 degradation and increased LC3-II conversion as compared to10 micomolar concentrations of the Tat-Beclin 1 compound; see,Shoji-Kawata et al. (2013), below.

The Tat-beclin 1 peptides could tolerate losing the two N-terminalbeclin 1 amino acids, but lost potency with the removal of the thirdN-terminal residue. The two required structural residues (F270 and F274)make up the N-terminal half of the beclin 1 peptide, forming a surfaceloop that may dictate activity. TAT-fusions corresponding to thisN-terminal half were tested alone and with the C-terminal half added intrans. Neither of these peptides, nor their combination, was able toinduce autophagy. This lack of activity suggests a requirement for somecomponent of the C-terminus in addition to the N-terminal aromaticstabilized loop. In the ECD structure, the C-terminal residues followingthe D (corresponding to S279 mutated for solubility) are largely buriedand unlikely to contribute directly to an interaction surface.Accordingly, omission of these five C-terminal residues from the beclin1 peptide yielded the most potent inducer of autophagy thus tested. Thesurface components of the minimal peptide include 1) the N-terminal V 2)the aromatic intervening NAT and 2) the C-terminal WH. These residuesare inferred to contribute to a binding surface in the peptide thatmimics the ECD and is required for induction of autophagy by thepeptide.

We used targeted mutagenesis of amino acids that are predicted to beimportant based on their substitution patterns during evolution [PMID8609628; 15037084]. This approach has been successfully applied todesign point mutants that modify the activity of functional sites [PMID20385837] and that selectively block function [PMID 16280323]. Wesimilarly use a series of three or four different mutations at eachposition to probe both conservative to non-conservative substitutionsbased on standard transition matrices [PMID 1438297], using mutations toresidues predicted to be less important as negative controls to map outefficiently the key binding residues in the minimal peptide. To enhancepotency and identify activity determinants we can increasingly perturbresidues through increasingly less conservative substitutions, forexample, V269 to I, L, M then A, or 1276 to V, L, F, etc. We also usescanning mutagenesis to systematically change each residue one at atime.

F270 and F274 function to restrict the conformational flexibility of theintervening residues and reduce the entropy cost of binding. We prepareda number of more chemically- stable linkers via standard solid-phasepeptide synthesis. For instance, replacement of F270 with a lysine andF274 with an aspartic or glutamic acid residue allows the synthesis ofmacrocyclic lactam peptides of incrementally-larger ring size. Even morechemically-stable linkages can be achieved through ring-closingmetathesis strategies, where terminal alkene handles derived fromcommercial non-natural amino acids offer a chemical handle forcyclization. This latter carbon-carbon based cyclization modality isoften called peptide “stapling”, and has been reviewed extensively (PMID22230563). An advantage of backbone “staples” is the reducedsusceptibility of carbon-carbon linkages to proteolytic cleavage.“Click” variants derived from [3+2] cycloaddition reactions may also beused for peptide cyclization (PMID 23064223).

These various cyclization strategies enforce the bioactive conformationof the beclin peptide, and can be used to increase the cellularpermeability relative to the uncyclized peptide. Peptide conformationcan significantly influence the ability of macromolecules to penetrateinto cells (PMID: 23133740). In particular, backbone modifications canhave dramatic impact on cellular permeability, potentially creatingopportunities for oral administration of cyclic peptides (PMID 22737969and PMID 12646037). Optionally combined with retro-inverso peptidestrategies to further limit amide hydrolysis (PMID 23382963), thesecompositions enable use the disclosed compounds as peptide therapeutics.

TABLE 1 Targeted mutagensis Peptide Sequence* SEQ ID NO ActivityWild-type (wt) VFNATFEIWHD SEQ ID NO: 03 wt Mutant 1 CFNATFEIWHDSEQ ID NO: 10 ~wt Mutant 2 VWNATFEIWHD SEQ ID NO: 11 >wt Mutant 3VFDATFEIWHD SEQ ID NO: 12 <<wt Mutant 4 VFNSTFEIWHD SEQ ID NO: 13 <<wtMutant 5 VFNACFEIWHD SEQ ID NO: 14 <wt Mutant 6 VFNATWEIWHDSEQ ID NO: 15 <wt Mutant 7 VFNATFDIWHD SEQ ID NO: 16 ~wt Mutant 8VFNATFELWHD SEQ ID NO: 17 >wt Mutant 9 VFNATFEIFHD SEQ ID NO: 18 ~wtMutant 10 VFNATFEIWYD SEQ ID NO: 19 ~wt Mutant 11 VFNATFEIWHESEQ ID NO: 20 ~wt Mutant 12 VWNATFELWHD SEQ ID NO: 21 ~wt Mutant 13VYNATFEIWHD SEQ ID NO: 22 <wt Mutant 14 VFNATFEVWHD SEQ ID NO: 23 ~wtMutant 15 VLNATFEIWHD SEQ ID NO: 24 ~wt Mutant 16 VFNATFEMWHDSEQ ID NO: 25 ~wt Mutant 17 VWNATFHIWHD SEQ ID NO: 26 >>wt Mutant 18VFNATFEFWHD SEQ ID NO: 27 ~wt Mutant 19 VFNATFEYWHD SEQ ID NO: 28 ~wtMutant 20 VFNATFERWHD SEQ ID NO: 29 ~wt Mutant 21  FNATFEIWHDSEQ ID NO: 30 >wt Mutant 25 VFNATFEIWH SEQ ID NO: 31 >wt Mutant 26 FNATFEIWH SEQ ID NO: 32 >>wt Mutant 27  WNATFHIWH SEQ ID NO: 33 >>wtMutant 28 VWNATFHIWH SEQ ID NO: 34 >>wt Mutant 29  WNATFHIWHDSEQ ID NO: 35 >>wt Mutant 30 DHWIHFTANWV SEQ ID NO: 36 >>wt *The 11amino acids PTD from HIV TAT and the diglycine linker are not listed;this is the same for all peptides. Mutant 17 and its derivatives are themost active, and activity is retained with the correspondingD-retro-inverso sequences (e.g. Mutant 30). Activity was measured asautophagy inducing activity, as described supra.

REFERENCES

[1] S. Shoji-Kawata, R. Sumpter, M. Leveno, G. R. Campbell, Z. Zou, L.Kinch, A. D. Wilkins, Q. Sun, K. Pallauf, D. MacDuff, C. Huerta, H. W.Virgin, J. B. Helms, R. Eerland, S. A. Tooze, R. Xavier, D. J. Lenschow,A. Yamamoto, D. King, O. Lichtarge, N. V. Grishin, S. A. Spector, D. V.Kaloyanova, B. Levine, Identification of a candidate therapeuticautophagy-inducing peptide, Nature, 494 201-206, 2013.

[2] A. Bryan, L. Joseph, J. A. Bennett, H. I. Jacobson, T. T. Andersen,Design and synthesis of biologically active peptides: a ‘tail’ of aminoacids can modulate activity of synthetic cyclic peptides, Peptides, 322504-2510.

[3] J. P. Rose, C. K. Wu, C. D. Hsiao, E. Breslow, B. C. Wang, Crystalstructure of the neurophysin-oxytocin complex, Nat Struct Biol, 3 (1996)163-169.

[4] K. J. Rosengren, R. J. Clark, N. L. Daly, U. Goransson, A. Jones, D.J. Craik, Microcin J25 has a threaded sidechain-to-backbone ringstructure and not a head-to-tail cyclized backbone, J Am Chem Soc, 125(2003) 12464-12474.

What is claimed is:
 1. An autophagy-inducing compound comprising anautophagy-inducing peptide comprising Beclin 1 residue 269 or 279 toBeclin 1 residue 278 or 279, wherein Beclin 1 residues 269-279 isVFNATFHIWHS (SEQ ID NO:01), or the D-retro-inverso sequence thereof,wherein the peptide is immediately N- and C-terminally flanked bymoieties R₁ and R₂, respectively, wherein up to six of said residues maybe substituted, R₁ and R₂ do not naturally flank the Beclin 1 peptide,and F270 and F274 are optionally substituted and optionally linked. 2.The compound of claim 1 wherein the peptide comprises a sequence,unsubstituted, selected from: Peptide Sequence SEQ ID NO Wild-type (wt)VFNATFEIWHD SEQ ID NO: 03; Mutant 1 CFNATFEIWHD SEQ ID NO: 10; Mutant 2VWNATFEIWHD SEQ ID NO: 11; Mutant 3 VFDATFEIWHD SEQ ID NO: 12; Mutant 4VFNSTFEIWHD SEQ ID NO: 13; Mutant 5 VFNACFEIWHD SEQ ID NO: 14; Mutant 6VFNATWEIWHD SEQ ID NO: 15; Mutant 7 VFNATFDIWHD SEQ ID NO: 16; Mutant 8VFNATFELWHD SEQ ID NO: 17; Mutant 9 VFNATFEIFHD SEQ ID NO: 18; Mutant 10VFNATFEIWYD SEQ ID NO: 19; Mutant 11 VFNATFEIWHE SEQ ID NO: 20;Mutant 12 VWNATFELWHD SEQ ID NO: 21; Mutant 13 VYNATFEIWHDSEQ ID NO: 22; Mutant 14 VFNATFEVWHD SEQ ID NO: 23; Mutant 15VLNATFEIWHD SEQ ID NO: 24; Mutant 16 VFNATFEMWHD SEQ ID NO: 25;Mutant 17 VWNATFHIWHD SEQ ID NO: 26; Mutant 18 VFNATFEFWHDSEQ ID NO: 27; Mutant 19 VFNATFEYWHD SEQ ID NO: 28; Mutant 20VFNATFERWHD SEQ ID NO: 29; Mutant 21 FNATFEIWHD SEQ ID NO: 30; Mutant 25VFNATFEIWH SEQ ID NO: 31; Mutant 26  FNATFEIWH SEQ ID NO: 32; Mutant 27 WNATFHIWH SEQ ID NO: 33; Mutant 28 VWNATFHIWH SEQ ID NO: 34; andMutant 29  WNATFHIWHD SEQ ID NO: 35,

or the D-retro-inverso sequence of the peptide.
 3. The compound of claim1, wherein R₁ comprises a transduction domain, a homing peptide, or aserum stabilizing agent.
 4. The compound of claim 1 wherein R₁ is a tatprotein transduction domain linked to the peptide through a diglycinelinker, particularly a diglycine-T-N linker.
 5. The compound of claim 1wherein R₂ is carboxyl or R₂ comprises an affinity tag or detectablelabel, particularly a fluorescent label.
 6. The compound of claim 1wherein: a) F270 and F274 are substituted with crosslinkable moietiesand/or linked, and each optionally comprises an additional a-carbonsubstitution selected from substituted, optionally hetero- lower alkyl,particularly optionally substituted, optionally hetero- methyl, ethyl,propyl and butyl; or b) F270 and F274 are substituted with homocysteinesconnected through a disulfide bridge to generate a ring and tail cyclicpeptide.
 7. The compound of claim 1 wherein the side chains of F270 andF274 are replaced by a linker: —(CH₂)_(n)ONHCOX(CH₂)_(m)—, wherein X isCH₂, NH or O, and m and n are integers 1-4, forming a lactam peptide;—CH₂OCH₂CHCHCH₂OCH₂—, forming an ether peptide; or—(CH₂)_(n)CHCH(CH₂)_(m)—, forming a stapled peptide.
 8. The compound ofclaim 1 wherein: 1 to 6 residues are alanine substituted; or the peptidecomprises at least one of substitutions: H275E and S279D; or the peptidecomprises one or more D-amino acids, L-β-homo amino acids, D-β-homoamino acids, or N-methylated amino acids; or the peptide comprises theD-retro-inverso sequence.
 9. The compound of claim 1 comprising theD-retro-inverso sequence: (SEQ ID NO: 08) RRQRRKKKRGY-GG-DHWIEFTANFV.


10. The compound of claim 1 wherein the peptide is acetylated, acylated,formylated, amidated, phosphorylated, sulfated or glycosylated.
 11. Thecompound of claim 1 comprising an N-terminal acetyl, formyl, myristoyl,palmitoyl, carboxyl or 2-furosyl group, and/or a C-terminal hydroxyl,amide, ester or thioester group.
 12. The compound of claim 1 wherein thepeptide is cyclized.
 13. The compound of claim 1 wherein the peptidecomprises the sequence VWNATFHIWHD (SEQ ID NO:26) or theD-retro-inverso- sequence thereof, DHWIHFTANWV (SEQ ID NO:30).
 14. Apharmaceutical composition comprising a compound of claim 1 in unitdosage, administrable form.
 15. A method of inducing autophagy,comprising administering to a person in need thereof an effective amountof a compound of claim 1.